CN103797027A - Variant polypeptide having homoserine acetyltransferase activity and microorganism expressing same - Google Patents

Abstract

The present invention relates to polypeptides modified to have high serine O-acetyltransferase activity, and more specifically, to provide variant polypeptides having high serine O-acetyltransferase activity by substituting an amino acid at position 111 of the starting methionine in polypeptides having high serine succinyltransferase activity.

Description

Modified polypeptides having homoserine acetyltransferase activity and microorganisms expressing them

Background technique

1. Field of invention

The present invention relates to a polypeptide modified to have homoserine acetyltransferase activity, a polynucleotide encoding it, a recombinant vector comprising the polynucleotide, a microorganism transformed with the recombinant vector, and a method for producing O-acetyl homoserine using the microorganism method.

2. Description of related technologies

Methionine is one of the essential amino acids in the body, and has been widely used as animal feed and food additives, as well as components of medical aqueous solutions and other raw materials for pharmaceutical products. Methionine acts as a precursor for choline (lecithin) and creatine, and is also used as a raw material for the synthesis of cysteine and taurine. Furthermore, it functions as a sulfur donor.

S-adenosyl-methionine is derived from L-methionine and is used as a methyl donor in the body, and it participates in the synthesis of various neurotransmitters in the brain. Methionine and/or S-adenosyl-L-methionine (SAM) have also been found to prevent lipid accumulation in the liver and arteries and to be effective in treating depression, inflammation, liver disease and muscle pain.

Methionine can be chemically or biologically synthesized for use in animal feed, food and pharmaceuticals.

In chemical synthesis, L-methionine is mainly produced by hydrolysis of 5-(β-methylmercaptoethyl)-hydantoin. However, chemically synthesized methionine has the disadvantage of being produced only as a mixed form of L-form and D-form.

Regarding the biosynthesis of L-methionine, U.S. Patent Publication No. US2005/0054060A1 describes the method of directly utilizing H ₂ S or CH ₃ SH without using cysteine, by modifying cystathionine for microbial production Ether synthase synthesizes homocysteine or methionine. In this method, a modified cystathionine synthase is directly introduced into cells to synthesize methionine according to the intracellular methionine synthesis process. However, there is a practical problem in that this method produces only a small amount of methionine because of the inhibitory effect caused by methionine synthesized by utilizing the intracellular methionine metabolic pathway, and H ₂ S or CH ₃ SH also causes Cytotoxicity.

In order to solve these problems, the present inventors have developed a two-step method (PCT/KR2007/003650) for converting an L-methionine precursor into L-methionine by an enzymatic reaction. This two-step approach can solve the problem of cytotoxicity of H ₂ S or CH ₃ SH and inhibition of the metabolic process of the produced L-methionine. In addition, this method is characterized by selectively producing only L-methionine instead of D-methionine and L-methionine in a mixed form and is very effective.

In this two-step process, O-succinyl homoserine and O-acetyl homoserine can be used as methionine precursors. During the conversion reaction of methionine, O-acetyl homoserine is superior to O-succinyl homoserine in terms of the productivity of the precursor to methionine ratio. Specifically, 0.91 moles of methionine can be produced from 1 mole of O-acetyl homoserine, while only 0.67 moles of methionine can be produced from 1 mole of O-succinyl homoserine. Therefore, the production cost of the final product methionine can be reduced by using O-acetylhomoserine as a methionine precursor, and the high productivity of O-acetylhomoserine is a key factor for the mass production of methionine.

Meanwhile, the use of O-acetyl homoserine or O-succinyl homoserine as a methionine precursor depends on the type of microorganism. In detail, microorganisms belonging to the genus Escherichia, Enterobacteria, Salmonella, and Bacillus generate from homoserine and Succinyl-coA produces O-succinyl-homoserine (Biochemistry.1999Oct26;38(43):14416-23), and belongs to the genus Corynebacterium, Leptospira, Deinococcus , Pseudomonas (Pseudomonas) and Mycobacterium (Mycobacterium) microorganisms produce O-acetyl homoserine (Journal of Bacteriology, Mar.2002, p.1277-1286).

Therefore, the biosynthesis of O-acetyl homoserine using microorganisms of the genus Escherichia for the production of recombinant proteins for experimental and industrial purposes requires the transfer of O-acetyl homoserine expressed by the introduction of metX, an exogenous gene. enzyme. However, there are problems related to consumers' negative attitude towards the introduction of exogenous genes into microorganisms used for producing food and proving the safety of the introduction of exogenous genes.

Therefore, the present inventors have made efforts to prepare a strain of the genus Escherichia that produces O-acetylhomoserine that is advantageous in terms of productivity without introducing a foreign gene. As a result, they found that by using a modified polypeptide prepared by substituting glutamic acid for the amino acid at position 111 of O-succinyl homoserine transferase from E. transferase activity, thus completing the present invention.

Contents of the invention

The object of the present invention is to provide a modified polypeptide, wherein the polypeptide having homoserine O-succinyltransferase activity is converted to have homoserine acetyltransferase activity.

Another object of the present invention is to provide polynucleotides encoding the above modified polypeptides.

Another object of the present invention is to provide a recombinant vector comprising a polynucleotide sequence operably linked to the above polynucleotide.

Another object of the present invention is to provide microorganisms comprising the above polynucleotides.

Yet another object of the present invention is to provide a microorganism transformed with a recombinant vector operably linked to the above polynucleotide.

Another object of the present invention is to provide a method for producing O-acetyl homoserine using a microorganism expressing a modified polypeptide having homoserine acetyltransferase activity.

Description of drawings

Figure 1 is a diagram showing a recombinant vector operably linked to a polynucleotide encoding a modified polypeptide according to the present invention;

Figure 2 shows the homology comparison of the primary amino acid sequence of homoserine O-succinyltransferase between E. coli variants;

Figures 3 and 4 show a homology comparison of the primary amino acid sequences of mutant homoserine O-succinyltransferases resistant to feedback regulation by methionine, among which the wild-type Homoserine O-succinyltransferase, Feedback-Resistant Mutant Homoserine O-succinyltransferases met10A and met11A, and Feedback-Resistant Mutant Homoserine O-succinyltransferases Disclosed in PCT Publication No. WO2005/108561 The primary amino acid sequence of is used for comparison; and

Figure 5 is a diagram showing the preparation of an FRT-one-step deletion cassette by overlapping PCR to replace the acs promoter with the pro promoter in the chromosome.

Detailed Description

In one aspect of achieving the above object, the present invention provides a modified polypeptide having homoserine O-acetyltransferase activity, the modified polypeptide has SEQ ID No.17 or an amino acid sequence at least 95% homologous thereto, wherein The amino acid at position 111 from the starting amino acid of the sequence, methionine, was replaced with glutamic acid.

As used herein, a polypeptide having homoserine O-succinyltransferase activity refers to a polypeptide having the activity of synthesizing O-succinyl homoserine from homoserine and succinyl-coA present in the methionine biosynthetic pathway, as follows The reaction scheme is displayed.

Homoserine+Succinyl-CoA->O-Succinyl-Homoserine

The polypeptide having homoserine O-succinyltransferase activity can be a recombinant polypeptide, which is derived from microorganisms of the genus Enterobacter, Salmonella, Pseudomonas, Bacillus or Escherichia, preferably, has A recombinant polypeptide having homoserine succinyltransferase activity from a microorganism of the genus Grease, and more preferably, a recombinant polypeptide having homoserine O-succinyltransferase activity from Escherichia coli.

In the present invention, the polypeptide having homoserine O-succinyltransferase activity may include a polypeptide having homoserine succinyltransferase activity, said polypeptide being made up of SEQ ID NO: 17 or its at least 95% homologous amino acid sequence , as long as it has the activity shown in the above reaction scheme.

In the examples of the present invention, the homology of amino acid sequences of homoserine O-succinyltransferases between different species of Escherichia coli was compared. As a result, there was less than 5% variation in homoserine O-succinyltransferase polypeptides between different species of Escherichia coli (that is, they had at least 95% homology), but homoserine O-succinyltransferase There was no significant difference in activity (Figure 2). These results indicate that polypeptides having 95% or more homology to the polypeptide of SEQ ID NO: 17 of the present invention also have the same homoserine O-succinyltransferase activity, which is obvious to those skilled in the art and determined by the present invention person visualization.

As used herein, the term "modified polypeptide" means a polypeptide having homoserine O-acetyltransferase activity by substituting a part of the amino acid sequence of the polypeptide having homoserine O-acetyltransferase activity, unlike wild type. That is, the modified polypeptide of the present invention refers to a modified polypeptide having the same activity as in the following reaction scheme, which has para-acetyl-CoA instead of succinyl by substituting a part of the amino acid sequence of the polypeptide having homoserine O-succinyltransferase activity - Substrate specificity of coA.

Homoserine+Acetyl CoA->O-Acetyl Homoserine

In the present invention, the above modified polypeptide may be a modified polypeptide, wherein at position 111 of a polypeptide having an amino acid sequence of SEQ ID NO: 17 or a polypeptide having a sequence homology of 95% or more to SEQ ID NO: 17 The amino acid was substituted with glutamic acid (SEQ ID NO.: 18), and the amino acid at position 112 of the polypeptide was further substituted with threonine (SEQ ID NO: 19) or histidine (SEQ ID NO: 20).

Further substitution of threonine or histidine for the amino acid leucine at position 112 was found to enhance homoserine acetyltransferase activity (Tables 2 and 3).

According to a preferred embodiment, the above modified polypeptide may be a polypeptide having any one of the amino acid sequences of SEQ ID NO: 18 to 20.

In an example of the present invention, a nucleus capable of expressing the amino acid glycine at position 111 of a homoserine succinyltransferase represented by SEQ ID NO: 39 was prepared in which glutamic acid was substituted for the amino acid glycine. The metA gene of Escherichia coli composed of a nucleotide sequence consists of a plasmid for a polypeptide encoded by - and a plasmid capable of expressing a polypeptide wherein the amino acid at position 112 is substituted with threonine or histidine in addition to the above substitutions (Example 2).

Further, the experimental examples of the present invention showed that only O-succinyl homoserine was produced by CJM2pCL_Pcj1_metA (wt) and CJM3pCL_Pcj1_metA (wt) transformed with a plasmid including the wild-type metA gene (SEQ ID NO: 39). In contrast, only O-acetyl homoserine was accumulated by the strain transformed with the plasmid including the gene encoding the modified polypeptide of the present invention (Experimental Example 2, Tables 2 and 3).

Therefore, a microorganism expressing the modified polypeptide of the present invention is advantageous because it is capable of producing O-acetylhomoserine as a methionine precursor capable of high-yield production without introducing a foreign gene for homoserine acetyltransferase activity.

In the present invention, the above modified polypeptide may be resistant to feedback regulation of methionine by substituting a part of amino acids of the polypeptide having homoserine succinyltransferase activity. That is, most of the activity of homoserine succinyltransferase is regulated by feedback inhibition of small amounts of methionine in the culture medium, and thus the modified polypeptides of the present invention may be resistant to feedback regulation of methionine for O-acetyl Mass production of homoserine.

In the present invention, amino acid substitutions that avoid feedback regulation of methionine can be performed according to the method disclosed in PCT Publication No. WO2008/127240. In detail, the feedback regulation of methionine can be avoided by making the following substitutions to the polypeptide having homoserine succinyltransferase activity: substitution of proline for the amino acid at position 29, substitution of glycine for the amino acid at position 114 substitution, substitution of serine to the amino acid at position 140, or a combination of one or more of the three amino acid substitutions. Preferably, two or more, and most preferably three amino acids are substituted.

According to a preferred embodiment, the modified polypeptide resistant to the feedback regulation of methionine can be a modified polypeptide having any amino acid sequence selected from the amino acid sequences of SEQ ID NO: 21 to 23.

In an embodiment of the present invention, amino acids at positions 29, 114 and 140 of the recombinant polypeptide encoded by the metA gene of Escherichia coli with homoserine succinyl transferase activity are replaced by proline, glycine and serine, respectively, to avoid Feedback regulation of methionine. In addition, a plasmid including a polynucleotide encoding a modified polypeptide having homoserine acetyltransferase activity prepared by substitution of glutamic acid for the amino acid at position 111 [pCL_Pcj1_metA#11(EL)] was constructed by [pCL_Pcj1_metA#11(ET)] prepared by substitution of amino acids at positions 111 and 112 by glutamic acid and threonine, and [ pCL_Pcj1_metA#11(EH)] (Example 3).

Further, the experimental examples of the present invention show that in a strain expressing a modified polypeptide resistant to feedback regulation by methionine, substitution of amino acids at positions 111 and 112 by glutamic acid and histidine produces The CJM2pCL_Pcj1_metA(#11)EH and CJM3pCL_Pcj1_metA(#11)EH strains showed high O-acetyl homoserine productivity of 11.1 g/L and 24.8 g/L, respectively, and these accumulations of O-acetyl homoserine were similar to those obtained by introducing Those of the exogenous homoserine acetyltransferase gene (Experimental Example 2, Tables 2 and 3).

In another aspect, the present invention provides a polynucleotide encoding a modified polypeptide or a recombinant vector comprising a polynucleotide sequence operably linked to the polynucleotide.

In the present invention, the above polynucleotide is a nucleotide polymer composed of nucleotide monomers covalently linked in a chain, and an example thereof is a DNA or RNA chain having a predetermined length or longer, and it is A polynucleotide encoding the above modified polypeptide.

In the present invention, the above polynucleotide may be a polynucleotide having any one of the nucleotide sequences of SEQ ID NO: 24 to 29.

As used herein, the above term "recombinant vector" is a means for expressing a modified polypeptide by introducing DNA into a host cell in order to prepare a microorganism expressing the modified polypeptide of the present invention, and known expression vectors such as plasmid vectors can be used , cosmid vectors and phage vectors. Vectors can be readily prepared by those skilled in the art according to any known method utilizing recombinant DNA technology.

In the present invention, the recombinant vector can be pACYC177, pACYC184, pCL1920, pECCG117, pUC19, pBR322 or pMW118 vector, and preferably pCL1920 vector.

The term "operably linked" means that the expression regulatory sequence is linked in a manner that regulates the transcription and translation of the polynucleotide sequence encoding the modified polypeptide, and includes that when the polynucleotide sequence is under the control of the regulatory sequence (including a promoter) The precise translation frame is maintained when expressed in such a way as to produce the modified polypeptide encoded by the polynucleotide sequence.

In still another aspect, the present invention provides microorganisms comprising polynucleotides encoding the above modified polypeptides and microorganisms transformed with recombinant vectors operably linked to the polynucleotides encoding the above modified polypeptides.

As used herein, the term "transformation" means a method of introducing a gene into a host cell for expression in the host cell. The transforming gene, if it is in a state expressed in the host cell, may be inserted into the chromosome of the host cell or may exist independently of the chromosome.

In addition, the gene includes DNA and RNA which are polynucleotides capable of encoding a polypeptide. The gene can be introduced in any type as long as it can be introduced into and expressed in the host cell. For example, the gene can be introduced into the host cell in the form of an expression cassette, which is a polynucleotide construct that includes all the elements for expressing the gene on its own. Typically, the expression cassette includes a promoter, transcription termination signal, ribosome binding site, and translation termination signal, which are operably linked to the gene. The expression cassette may be a type of expression vector capable of self-replication. The above genes can also be introduced into host cells by themselves or in the form of polynucleotide constructs so as to be operably linked to sequences required for expression in the host cells.

The above microorganisms are prokaryotic or eukaryotic microorganisms capable of expressing a modified polypeptide by including a polynucleotide encoding a modified polypeptide or by transforming a recombinant vector that is operably linked to a polynucleotide encoding a modified polypeptide, and for example, it may belong to The following microorganisms: Escherichia, Bacillus, Aerobacter, Serratia, Providencia, Erwinia, Schizosaccharomyces, Enterobacter, Zygomyces, Hook Leptospirosis, Deinococcus, Pichia, Kluyveromyces, Candida, Hansenula, Debaria, Mucor, Torulopsis, Methylobacterium ( methylobacter), Salmonella, Streptomyces, Pseudomonas, Brevibacterium, or Corynebacterium.

In the present invention, the microorganism expresses a polypeptide having homoserine O-succinyltransferase activity. For example, it may be a microorganism belonging to the genus Bacillus, Escherichia, Enterobacter or Salmonella, preferably a microorganism belonging to the genus Escherichia, and more preferably Escherichia coli.

In an embodiment of the present invention, Escherichia coli CJM2pCL_Pcj1_metAEL, CJM2pCL_Pcj1_metAET and CJM2pCL_Pcj1_metAEH strains (Example 2 and Experimental Example 2) transformed with a recombinant vector comprising a polynucleotide encoding the modified polypeptide of the present invention were prepared, and the use of Escherichia coli CJM2pCL_Pcj1_metA(#11)EL, CJM2pCL_Pcj1_metA(#11) transformed by the recombinant vector of the polynucleotide that is resistant to the feedback regulation of methionine and has the modified polypeptide of homoserine O-acetyltransferase activity of the present invention ) ET and CJM2pCL_Pcj1_metA (#11) EH strains (Example 3 and Experimental Example 2). Among the above strains, the CJM2pCL_Pcj1_metA(#11)EL, CJM2pCL_Pcj1_metA(#11)ET and CJM2pCL_Pcj1_metA(#11)EH strains were named CA05-0546, CA05-0547 and CA05-0548, respectively, and were released on December 14, 2010 It was deposited at the Korean Culture Center of Microorganism and assigned accession numbers KCCM11145P, KCCM11146P, and KCCM11147P, respectively.

The present invention provides a modified polypeptide having homoserine O-acetyltransferase activity, wherein a part of the amino acid sequence of the polypeptide having homoserine O-succinyltransferase activity is replaced. Therefore, it is advantageous because when the modified polypeptide of the present invention is expressed in a microorganism expressing a polypeptide having only homoserine O-succinyltransferase activity, a polypeptide having homoserine O-acetyltransferase activity can be expressed , without introducing a foreign gene encoding homoserine O-acetyltransferase such as metX.

In the present invention, the above microorganisms may be additionally modified to have enhanced acetyl-CoA synthetase activity or pantothenate kinase activity resistant to feedback inhibition of CoA accumulation to produce a large amount of O-acetyl Homoserine microorganisms.

In the present invention, acetyl-CoA synthetase and pantothenate kinase are derived from various microorganisms, and genes encoding proteins having these activities are generally referred to as acs and coaA, respectively.

In the present invention, the enhancement of acetyl-CoA synthetase activity can be realized by modifying the promoter region and the nucleotide sequence of the 5'-UTR region of the acs gene encoding acetyl-CoA synthetase to enhance gene expression, and the activity of the protein can be achieved by It is enhanced by introducing mutations in the ORF region of the corresponding gene, and the protein expression level can be enhanced by introducing an extra copy of the corresponding gene on the chromosome or by introducing the corresponding gene with its own promoter or enhanced other promoters into the strain.

More specifically, acetyl-CoA synthetase activity can be enhanced by substitution of activity-enhanced promoters, induction of promoter mutations for enhanced activity, or increased gene copy number, and thus, the present invention provides improved O-acetyl homoserine A method of productivity, and E. coli prepared by the method. As an alternative to an activity-enhanced promoter, pTac, pTrc, pPro, pR, and pL known to have enhanced activity can be used.

According to a preferred embodiment, the present invention provides O-acetyl homoserine-producing strains in which the acs gene involved in the biosynthesis of acetyl-CoA is overexpressed by replacing its promoter with a constitutive expression promoter, pro promoter . The pro promoter can be part or all of SEQ ID NO:30.

The present invention further provides a microorganism introduced with a modified pantothenate kinase resistant to feedback inhibition of CoA accumulation in the CoA biosynthetic pathway. More specifically, the amino acid arginine at position 106 in the amino acid sequence of pantothenate kinase was replaced by alanine (SEQ ID NO: 40) so that it became resistant to feedback inhibition of CoA accumulation, which resulted in O - Improvement of acetyl homoserine productivity.

In the present invention, the above microorganisms may be microorganisms selected from the group consisting of genes encoding phosphoenolpyruvate carboxylase (ppc), genes encoding aspartate aminotransferase (aspC), and genes encoding aspartate semialdehyde The copy number of one or more genes of the hydrogenase gene (asd) is increased, or the promoter of the gene is replaced by an activity-enhanced promoter, or is mutated to have enhanced activity.

In the present invention, a series of enzymes have the activity of synthesizing O-acetyl homoserine from phosphoenolpyruvate, as shown in the following reaction schemes. Therefore, the accumulation of O-acetyl homoserine in cells can be induced by enhancing the expression of genes with these activities.

Phosphoenolpyruvate carboxylase (ppc)

Phosphoenolpyruvate + H ₂ O + CO ₂ <-> Oxaloacetate + Phosphate

Aspartate aminotransferase (aspC)

Oxaloacetate + glutamic acid <-> aspartic acid + a-ketoglutarate

Aspartokinase (thrA)

Aspartate+ATP<->Aspartyl-4-phosphate+ADP

Aspartate semialdehyde dehydrogenase (asd)

Aspartyl-4-phosphate+NADPH<->aspartate-semialdehyde+phosphate+NADP+homoserine dehydrogenase (thrA)

Aspartic acid-semialdehyde+NADPH<->homoserine

In this reaction scheme, the thrA gene encoding the bifunctional enzyme aspartokinase/homoserine dehydrogenase was previously enhanced by the relief of feedback inhibition of the CJM2 strain in Experimental Example 2, and the remaining three enzymes Enhancement can be achieved by increasing the copy number of the gene, replacing the promoter of the above gene with an activity-enhanced promoter, or inducing mutations in the promoter to enhance activity.

As used herein, the term "copy number increase" means additional introduction of a desired gene into a chromosome or by introduction of a plasmid with a gene encoding the corresponding enzyme.

In an example of the present invention, the CJM2-AP strain was prepared by deleting the acs promoter of the metA and metB-deleted CJM2 strain and replacing it with the pro promoter, and subsequently transformed to have feedback-resistant coaA in order to produce The CJM2-AP/CO strain with increased acetyl-CoA concentration was followed by the preparation of the CJM3 strain with two copies of the three genes ppc, aspC and asd. Hereinafter, the pCL_Pcj1_metA#11(EL), pCL_Pcj1_metA#11(EH) and pCL_Pcj1_metA#11(ET)-introduced CJM3 strains were named CA05-0578, CA05-0579 and CA05-0580, respectively, and were established in December 2011 It was deposited at the Korean Collection of Microorganisms on the 12th, and assigned accession numbers KCCM11228P, KCCM11229P, and KCCM11230P, respectively (Experimental Example 2).

In yet another aspect, the present invention provides a method for producing O-acetyl homoserine comprising the steps of: culturing a microorganism comprising a polynucleotide encoding a modified polypeptide or using a recombinant polynucleotide operably linked to a polynucleotide encoding a modified polypeptide A vector-transformed microorganism, and O-acetyl homoserine produced during the cultivation of the above microorganism is obtained.

In the present invention, the production of O-acetyl homoserine using a microorganism expressing a modified polypeptide can be carried out using suitable medium and conditions known in the art. It is well understood by those skilled in the art that culture methods can be readily adapted to the strain of choice.

Examples of culture methods include, but are not limited to, batch, continuous, and fed-batch culture. The medium used in the cultivation must satisfy the cultivation conditions of the specific strain.

The substratum used in the present invention can comprise any one carbon source in sucrose, glucose, glycerol and acetic acid or its combination, and the nitrogen source used is example with organic nitrogen source and inorganic nitrogen source or its combination, described organic nitrogen Sources such as peptone, yeast extract, beef extract, malt extract, corn steep liquor, and soybean meal, and inorganic nitrogen sources such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate.

The medium may include monopotassium phosphate, dipotassium phosphate, and the corresponding sodium-containing salts as sources of phosphate. The medium may also include metal salts, such as magnesium sulfate or ferric sulfate. In addition, amino acids, vitamins and suitable precursors may also be added. Media or precursors can be added to the culture in batch or continuous mode. The pH of the culture can be adjusted by appropriately adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid during the cultivation, and can be suppressed by using an antifoaming agent such as fatty acid polyethylene glycol ester during the cultivation. Foam is produced.

To maintain the aerobic conditions of the culture, oxygen or an oxygen-containing gas can be injected into the culture. To maintain anaerobic and microaerophilic conditions, no gas can be injected or nitrogen, hydrogen or carbon dioxide can be injected. The temperature of the culture may be from 27°C to 37°C, and preferably from 30°C to 35°C. The cultivation period can be continued as long as the desired substance is produced, and is preferably continued for 10 to 100 hours.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. However, these examples are for illustrative purposes only, and the present invention is not intended to be limited by these examples.

Example 1: Including homoserine O-succinyltransferase and homoserine O-acetyltransferase Enzyme plasmid construction

Using the chromosome of Escherichia coli W3110 strain (accession number ATCC9637) purchased from the American Type Culture Collection (American Type Culture Collection) as a template and primers of SEQ ID NO: 1 and SEQ ID NO: 2 to implement PCR to amplify the The metA gene of serine O-succinyltransferase.

The primers used in PCR were prepared based on the sequence of the E. coli chromosome of NC_000913 registered in NIH Gene Bank, and the primers of SEQ ID NO:1 and SEQ ID NO:2 had EcoRV and HindIII restriction sites, respectively.

<SEQ ID NO:1>

5'AATTGATATCATGCCGATTCGTGTGCCGG3'

<SEQ ID NO:2>

5'AATTAAGCTTTTAATCCAGCGTTGGATTCATGTG3'

PCR was performed using the chromosome of Deinococcus radiodurans as a template and primers of SEQ ID NO:3 and SEQ ID NO:4 to amplify the metX gene encoding homoserine O-acetyltransferase (SEQ ID NO:44) . The primers of SEQ ID NO:3 and SEQ ID NO:4 have EcoRV and HindIII restriction sites, respectively.

<SEQ ID NO:3>

5'AATTGATATCATGACCGCCGTGCTCGC3'

<SEQ ID NO:4>

5'AATTAAGCTTTCAACTCCTGAGAAACGCCCC3'

PCR was carried out under the following conditions: denaturation at 94°C for 3 minutes; 25 cycles consisting of: denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds and polymerization at 72°C for 5 minutes; and at 72°C Polymerization was carried out at °C for 7 minutes.

After treatment with restriction enzymes, EcoRV and HindIII, respectively, the obtained PCR products were cloned into pCL1920 plasmid containing cj1 promoter (KR2006-0068505). Escherichia coli DH5α was transformed with the cloned plasmid, and the transformed Escherichia coli DH5α was selected on an LB plate containing spectinomycin at 50 μg/ml to obtain the plasmid. The obtained plasmids were named pCL_Pcj1_metA and pCL_Pcj1_metXdr, respectively.

Embodiment 2: the construction of the modified polypeptide with homoserine O-acetyltransferase activity

Using the pCL_Pcj1_metA plasmid prepared in Example 1 as a template and a site-directed mutagenesis kit (Stratagene, USA) (G111E), the amino acid glycine at position 111 of the O-succinyltransferase was replaced by glutamic acid (Glu) ( Gly). The sequences of the primers used are as follows:

<SEQ ID NO:5>

5'ttgtaactggtgcgccgctggaactggtggggtttaatgatgtc3'

<SEQ ID NO:6>

5'gacatcattaaaccccaccagttccagcggcgcaccagttacaa3'

The constructed plasmid containing the mutant G111E metA gene was named pCL_Pcj1_metA(EL).

In addition, the amino acid glycine (Gly) at position 111 of O-succinyltransferase is replaced by glutamic acid (Glu), and the amino acid leucine at position 112 of O-succinyltransferase is replaced by threonine (L112T) Or histidine (L112H) substitution. At this time, the sequences of the primers used are as follows:

Replace Leucine with Threonine

<SEQ ID NO:7>

5'tgtaactggtgcgccgctggaaaccgtggggtttaatgatgtcg3'

<SEQ ID NO:8>

5'cgacatcattaaaccccacggtttccagcggcgcaccagttaca3'

replace leucine with histidine

<SEQ ID NO:9>

5'tgtaactggtgcgccgctggaacatgtggggtttaatgatgtcg3'

<SEQ ID NO:10>

5'cgacatcattaaaccccacatgttccagcggcgcaccagttaca3'

Among the constructed plasmids, the plasmid containing the metA gene in which the amino acid glycine at position 111 was replaced with glutamic acid and the amino acid leucine at position 112 was replaced with threonine was named pCL_Pcj1_metA(ET). Likewise, a plasmid containing the metA gene in which the amino acid glycine at position 111 was replaced by glutamic acid and the amino acid leucine at position 112 was replaced by histidine was named pCL_Pcj1_metA(EH).

Embodiment 3: structure of anti-feedback modified polypeptide with homoserine O-acetyltransferase activity establish

Using the pCL_Pcj1_metA plasmid prepared in Example 1 as a template, a metA gene (metA#11) having feedback regulation resistance to methionine was constructed in the same manner as in Example 2. Specifically, according to the method disclosed in PCT Publication No. WO2008/127240, serine, glutamic acid, and phenylalanine at positions 29, 114, and 140 of O-succinyltransferase were respectively converted from proline (S29P), glycine (E114G) and serine (F140S) substitutions. The sequences of the primers used are as follows.

Replace Serine with Proline

<SEQ ID NO:11>

5'ATGACAACTTCTCGTGCGCCTGGTCAGGAAATTCG3'

<SEQ ID NO:12>

5'CGAATTTCCTGACCAGGCGCACGAGAAGTTGTCAT3'

Glycine instead of Glutamate

<SEQ ID NO:13>

5'CGCCGCTGGGCCTGGTGGGGTTTAATGATGTCGCT3'

<SEQ ID NO:14>

5'AGCGACATCATTAAACCCCCACCAGGCCCAGCGGCG3'

Serine instead of Phenylalanine

<SEQ ID NO:15>

5'CACGTCACCTCGACGCTGAGTGTCTGCTGGGCGGT3'

<SEQ ID NO:16>

5'ACCGCCCAGCAGACACTCAGCGTCGAGGTGACGTG3'

Each mutation was then introduced to construct a plasmid containing the metA(#11) gene with three mutations, which was named pCL_Pcj1_metA#11.

Subsequently, a plasmid expressing a polypeptide having the same mutation as the homoserine O-acetyltransferase activity-modified polypeptide of Example 2 using the prepared pCL_Pcj1_metA#11 plasmid as a template was constructed.

Among the constructed plasmids, the plasmid containing the metA#11 gene was named pCL_Pcj1_metA#11(EL), wherein the amino acid glycine at position 111 was replaced by glutamic acid, and the plasmid containing the metA#11 gene was named pCL_Pcj1_metA#11( ET), wherein the amino acid glycine at position 111 was replaced by glutamic acid and the amino acid leucine at position 112 was replaced by threonine, and the plasmid containing the metA#11 gene was named pCL_Pcj1_metA#11(EH), where position The amino acid glycine at position 111 was replaced by glutamic acid and the amino acid leucine at position 112 was replaced by histidine.

Experimental Example 1: Escherichia coli homoserine succinyltransferase and feedback-resistant Escherichia coli Homology comparison among homoserine succinyltransferases

Comparison of homoserine O-succinyl of Escherichia coli O9:H4 (strain HS), Escherichia coli O139:H28 (strain E24377A) and Escherichia coli O157:H7 (strain ATCC8739) variants using the program CLC Main Workbench (CLC bio, Denmark) Primary amino acid sequence of transferase [SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43 in that order].

As shown in Figure 2, less than 5% variation was observed in the primary amino acid sequence of the homoserine O-succinyltransferase of the E. coli variants (Figure 2).

The primary amino acid sequences of mutant homoserine O-succinyltransferases resistant to feedback regulation by methionine were also compared using the above procedure. For comparison, the wild-type homoserine O-succinyltransferase disclosed in PCT Publication No. WO2008/127240, the anti-feedback regulation mutant homoserine O-succinyltransferases met10A and met11A, and the homoserine O-succinyltransferase disclosed in PCT Publication No. WO2005/108561 were used. Primary amino acid sequence of a feedback-resistant mutant homoserine O-succinyltransferase.

As shown in Figures 3 and 4, less than 5% variation was observed in the primary amino acid sequence of mutant homoserine O-succinyltransferases resistant to feedback regulation by methionine (Figures 3 and 4).

These results indicate that homoserine O-succinyltransferase polypeptides present in Escherichia coli have 95% or more homology among them, and that there is no large difference in homoserine succinyltransferase activity even with less than 5% sequence difference.

Experimental Example 2: Substrates between modified polypeptides with homoserine acetyltransferase activity Comparison of Specificity and Activity of Species

2-1: Preparation of test strains

2-1-1) Deletion of metA and metB genes

In order to compare the activity of modified polypeptides producing excess O-acetyl homoserine, a strain that accumulates homoserine and has a deletion of O-acetyl homoserine utilization was prepared. The metA and metB gene-deleted strains were prepared by the method of Examples 1-1 to 1-4 described in published patent EP2108693A2 based on the threonine-producing strain FTR2533 (KCCM10541) described in PCT/KR2005/00344. This strain was named CJM2. CJM2 is a strain that accumulates a large amount of homoserine and produces O-acetyl homoserine or O-succinyl homoserine depending on the introduced gene.

2-1-2) Replacement of acs promoter

In order to produce excess O-acetyl homoserine, it is necessary to promote the production of homoserine and acetyl-CoA. First, in order to facilitate the supply of acetyl-CoA, the promoter of the acs (acetyl-CoA synthetase) gene was replaced by the constitutive pro promoter of SEQ ID NO: 30 in order to induce constitutive overexpression of the desired gene. To replace the promoter, a modified FRT-one-step PCR (PNAS (2000) vol. 97:6640-6645) was performed. To prepare the cassette as shown in Figure 5, a chloramphenicol-resistant FRT cassette derived from pKD3 (PNAS (2000) vol. 97:6640-6645) was subjected to PCR using SEQ ID NO: 31 and SEQ ID NO: 33, and The pro promoter region was subjected to PCR using SEQ ID NO:32 and SEQ ID NO:34. The two PCR products were subjected to overlapping PCR to make a single cassette (acs promoter deleted-pro promoter replaced cassette) (Nucleic Acids Res. 1988 August 11;16(15):7351-7367). PCR was performed under the following conditions: 30 cycles consisting of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute.

<SEQ ID NO:31>

5'

AGGGGCTTCATCCGAATTGCGCCATTGTTGCAATGGCGGTGCTGGAGCTGCTTCGAAGTTC3'

<SEQ ID NO:32>

5'

GATATTCATATGGACCATGGCTCGAGCATAGCATTTTTATCC3'

<SEQ ID NO:33>

5'

GGATAAAAATGCTATGCTCGAGCCATGGTCCATATGAATATC3'

<SEQ ID NO:34>

5'

CGATGTTGGCAGGAATGGTGTGTTTGTGAATTTGGCTCATATGTACCTTTTCTCCTCTTTA3'

The resulting PCR product was electrophoresed on a 1.0% agarose gel, and DNA was subsequently purified from a band of about 1.2 kbp. The recovered DNA fragment was electroporated into the CJM2 strain previously transformed with the pKD46 vector (PNAS (2000) vol. 97:6640-6645). Before electroporation, the CJM2 strain transformed with pKD46 was cultured at 30°C in LB medium containing 100 μg/L ampicillin and 5 mM L-arabinose until OD600 reached 0.6. Subsequently, the cultured strains were washed once with sterilized distilled water and twice with 10% glycerol. Electroporation was performed at 2500V. The recovered strain was streaked on an LB plate medium containing 25 μg/L of chloramphenicol, followed by culturing overnight at 37°C. Subsequently, strains showing resistance to chloramphenicol were thus selected.

PCR was performed using the selected strain as a template and the same primers under the same conditions. Deletion of the acs promoter and substitution of the pro promoter were identified by confirming a 1.2 kb size gene on a 1.0% agarose gel. This strain was then transformed with pCP20 vector (PNAS (2000) vol.97:6640-6645) and cultured in LB medium. The final acs promoter deletion and pro promoter replacement strains were constructed in which the gene size was reduced to 150 bp by PCR on 1.0% agarose gel under the same experimental conditions, and the deletion of the chloramphenicol marker gene was confirmed. The constructed strain was named CJM2-AP.

2-1-3) Alternative anti-feedback coaA

To prepare a CJM2-AP strain with feedback-resistant coaA, PCR was performed using w3110gDNA as a template and primers of SEQ ID NO: 35 and SEQ ID NO: 36 containing an EcoRI restriction site in order to obtain a coaA gene encoding pantothenate kinase . High-fidelity DNA polymerase PfuUltra ^™ (Stratagene) was used as the polymerase, and PCR was carried out under the following conditions: consisting of denaturation at 96°C for 30 seconds; annealing at 50°C for 30 seconds; and polymerization at 72°C for 2 minutes 30 cycles.

After the obtained coaA gene and pSG76C plasmid (Journal of Bacteriology, July 1997, 4426-4428) were treated with restriction enzyme EcoRI, they were ligated to each other. Escherichia coli DH5 was transformed with the constructed plasmid, and then, the transformed Escherichia coli DH5α was selected on an LB plate medium containing 25 μg/ml of chloramphenicol to obtain pSG-76C-coaA.

<SEQ ID NO:35>

5'ATGAGTATAAAAAGAGCAAAC3'

<SEQ ID NO:36>

5'TTATTTGCGTAGTCTGACC3'

pSG-76C-coaA (R106A) was constructed by site-directed mutagenesis (Stratagene, USA) utilizing the obtained pSG-76C-coaA and the primers of SEQ ID NO: 37 and SEQ ID NO: 38.

<SEQ ID NO:37>

5'GGAAAAGTACAACCGCCgccGTATTGCAGGCGCTATT3'

<SEQ ID NO:38>

5'AATAGCGCCTGCAATACggcGGCGGTTGTACTTTTTCC3'

The CJM2-AP strain was transformed with the pSG76C-coaA (R106A) plasmid, and cultured in LB-Cm (yeast extract 10g/L, NaCl 5g/L, tryptone 10g/L, chloramphenicol 25μg/L) medium to Colonies resistant to chloramphenicol were selected. Selected transformants were strains in which pSG76c-coaA(R106A) was originally inserted into the coaA region of the genome.

The coaA(R106A) gene-inserted strain was transformed with the pASceP vector (Journal of Bacteriology, July 1997, 4426-4428) expressing the restriction enzyme I-SceI that cuts the I-SceI site present in pSG76c, followed by transformation in LB -Ap (yeast extract 10g/L, NaCl 5g/L, tryptone 10g/L, ampicillin 100μg/L) to select strains. The coaA gene was amplified from the selected strain using primers of SEQ ID NO:35 and SEQ ID NO:36, and the substitution of coaA(R106) in the amplified gene was confirmed by macrogen sequencing service (Korea) (Nucleic Acids Research, 1999 , Vol.27, No.224409-4415). The prepared strain was named CJM2-AP/CO. The CJM2-AP/CO strain is a strain with increased concentrations of homoserine and acetyl-CoA.

2-1-4) Increase in the copy number of key genes in the homoserine biosynthesis pathway

Even if the CJM2 or CJM2-AP/CO strain is a strain producing excess homoserine, the copy numbers of the three genes ppc, aspC and asd were increased to further improve homoserine productivity. The pSG76c-2ppc, pSG76c-2aspC, and pSG76c-2asd plasmids were constructed using the method described in Examples <1-1> to <1-3> of Publication Patent No. KR2011-0023703, and the plasmids were introduced into the CJM2-AP/CO strain to Strains having two copies of the three genes were prepared by the method of Example <1-5>. The prepared strain was named CJM3. CJM3 is a strain that accumulates a large amount of homoserine compared with the CJM2 strain and produces O-acetyl homoserine or O-succinyl homoserine depending on the introduced plasmid.

2-2: Experimental method and experimental results

Two strains CJM2 and CJM3 were prepared as competent cells, and nine kinds of plasmids pCL_Pcj1_metX, pCL_Pcj1_metA, pCL_Pcj1_metA(EL), pCL_Pcj1_metA(EH), pCL_Pcj1_metA(ET), pCL_Pcj1_metA#11, pCL_Pcj1_metA#11(EL), pCL_APcj (EH) and pCL_Pcj1_metA#11(ET) were introduced into the competent cells by electroporation, respectively.

Among them, the CJM2 strains into which pCL_Pcj1_metA#11(EL), pCL_Pcj1_metA#11(EH) and pCL_Pcj1_metA#11(ET) were introduced were named CA05-0546, CA05-0547 and CA05-0548, respectively. They were deposited at the Korean Collection of Microorganisms on December 14, 2010, and assigned accession numbers KCCM11145P, KCCM11146P, and KCCM11147P, respectively.

Further, the CJM3 strains into which pCL_Pcj1_metA#11(EL), pCL_Pcj1_metA#11(EH) and pCL_Pcj1_metA#11(ET) were introduced were named CA05-0578, CA05-0579 and CA05-0580, respectively. They were deposited at the Korean Collection of Microorganisms on December 12, 2011, and assigned accession numbers KCCM11228P, KCCM11229P, and KCCM11230P, respectively.

Thereafter, a flask test was performed to compare the type and productivity of methionine precursors produced by each of the strains introduced with 9 types of plasmids. In the beaker test, after streaking each strain on LB plates and culturing them in a 31°C incubator for 16 hours, a single colony was inoculated in 3 ml of LB medium, and then cultured in a 200rpm/31°C incubator for 16 hours. Hour.

25 ml of the methionine precursor production medium of Table 1 was put into a 250 ml flask, and per flask, 500 μl of culture broth was added thereto. Subsequently, the flask was incubated in a 200 rpm/31° C. incubator for 40 hours, and the type and productivity of methionine precursor produced by each of the plasmid-introduced strains were compared by HPLC. The results are shown in Table 2 (results for CJM2-type strains) and Table 3 (results for CJM3-type strains).

[Table 1]

components Concentration (per liter) glucose 70g ammonium sulfate 25g KH ₂ PO ₄ 1g MgSO ₄ 7H ₂ O 0.5g FeSO ₄ 7H ₂ O 5mg MnSO ₄ 8H ₂ O 5mg _ZnSO4 5mg calcium carbonate 30g Yeast extract 2g Methionine 0.3g threonine 1.5g

[Table 2]

[table 3]

As shown in Tables 2 and 3, only O-succinyl homoserine was produced by pCL_Pcj1_metA (wt) including the wild-type metA gene, but only O-acetyl homoserine was accumulated by the strain including the three mutated metA genes of the present invention . That is, the homoserine succinyltransferase activity of the polypeptide is modified to homoserine acetyltransferase activity by substituting its amino acid.

Further, among the three mutants of CJM3-type strains, the strain prepared by substituting glutamic acid for the amino acid at position 111 (EL) produced 2.1 g/L of O-acetyl homoserine, while the additional The strain (EH) prepared with an acid substitution of the amino acid at position 112 produced 3.2 g/L of O-acetyl homoserine, which was the highest yield of O-acetyl homoserine.

A strain expressing a modified polypeptide having homoserine acetyltransferase activity resistant to feedback regulation by methionine also showed the same result. Specifically, the strain introducing the metA#11(EH) gene, which is resistant to feedback regulation of methionine and replaces amino acids at positions 111 and 112 with glutamic acid and histidine, produced the largest amount of O- Acetyl homoserine (24.8 g/L), indicating that it accumulated O-acetyl homoserine at a level similar to that of the strain introduced with the exogenous homoserine acetyltransferase gene (CJM3pCL_Pcj1_metX, 23.7 g/L).

Invention effect

According to the present invention, O-acetyl homoserine can be produced from homoserine without introducing a foreign gene into a microorganism expressing an enzyme that converts homoserine into O-succinyl homoserine, and the above O-acetyl homoserine can be used as Precursor for the production of methionine. Therefore, when the present invention is applied to the production of methionine for use in foods, it is advantageous because it is possible to solve consumers' anxiety and negative attitude towards the introduction of foreign genes and to provide safety for the introduction of foreign genes question of evidence.

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Claims (18)

1. A modified polypeptide with homoserine O-acetyltransferase activity, said modified polypeptide has SEQ ID NO: 17 or an amino acid sequence at least 95% homologous thereto, wherein the starting amino acid methionine from said sequence The amino acid at position 111 was replaced with glutamic acid. 2. The modified polypeptide according to claim 1, wherein the amino acid at position 112 of the polypeptide is further substituted with threonine or histidine. 3. The modified polypeptide according to claim 1 or 2, wherein the modified polypeptide has any one of the amino acid sequences of SEQ ID NO: 18 to 20. 4. The modified polypeptide of claim 1, wherein the modified polypeptide exhibits resistance to feedback regulation by methionine by amino acid substitution. 5. The modified polypeptide according to claim 4, wherein the amino acid is substituted with proline at position 29, glycine at position 114, serine at position 140 or a combination of one or more thereof. 6. The modified polypeptide according to claim 5, wherein the amino acid at position 112 of the polypeptide is further substituted with threonine or histidine. 7. The modified polypeptide according to claim 5 or 6, wherein the modified polypeptide has any one of the amino acid sequences of SEQ ID NO:21 to 23. 8. A polynucleotide encoding the modified polypeptide of claim 1. 9. The polynucleotide according to claim 8, wherein the polynucleotide has any one of the nucleotide sequences of SEQID NO:24 to 29. 10. A recombinant vector comprising a polynucleotide sequence operably linked to the polynucleotide of claim 8. 11. A microorganism comprising the polynucleotide of claim 8. 12. The microorganism according to claim 11 , wherein the microorganism is additionally modified to have enhanced acetyl-CoA synthetase activity compared to endogenous acetyl-CoA synthetase activity, or the microorganism is additionally modified to Has pantothenate kinase activity that is resistant to feedback inhibition of CoA accumulation. 13. The microorganism according to claim 11, wherein the copy number of one or more genes selected from the group consisting of the gene (ppc) encoding phosphoenolpyruvate carboxylase, the gene encoding aspartate aminotransferase ( aspC) and the gene encoding aspartate semialdehyde dehydrogenase (asd), or the promoter of said gene is replaced by an activity-enhanced promoter or mutated to have enhanced activity. 14. A microorganism transformed with the recombinant vector of claim 10. 15. The microorganism according to claim 14, wherein the microorganism belongs to the genus Escherichia. 16. The microorganism according to claim 15, wherein the microorganism is Escherichia coli (E. coli). 17. The microorganism according to claim 16, wherein said microorganism is deposited under accession numbers KCCM11145P, KCCM11146P, KCCM11147P, KCCM11228P, KCCM11229P or KCCM11230P. 18. A method for producing O-acetyl homoserine, comprising culturing the microorganism according to any one of claims 11 to 17; and obtaining O-acetyl homoserine produced during the cultivation of the microorganism.

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